Crystalline forms of an nk-1 antagonist

ABSTRACT

The present invention is related to crystalline forms of 2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamide which is an NK-1 antagonist useful in the treatment of induced vomiting and other disorders.

FIELD OF THE INVENTION

The present invention is related to crystalline forms of2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewhich is an NK-1 antagonist useful in the treatment of induced vomitingand other disorders.

BACKGROUND OF THE INVENTION

The compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidehaving Formula I:

is an antagonist of NK-1 useful in the treatment of various disordersincluding motion sickness and induced vomiting. The compound of FormulaI, as well as its preparation and use, have been described in U.S. Pat.No. 6,297,375, which is incorporated herein by reference in itsentirety.

For the development of a drug, it is typically advantageous to employ aform of the drug having desirable properties with respect to itspreparation, purification, reproducibility, stability, bioavailability,and other characteristics. U.S. Pat. No. 6,297,375 discloses a solidfree base form of the compound of Formula I in Example 14 (g) which isisolated by flash chromatography to yield the compound as “whitecrystals” with a melting point of 155-157° C. The example does notreport the crystalline peaks for this free base. The example also doesnot report whether this crystalline form of the free base was solvatedor hydrated. The compound was subsequently crystallized as the HCl salt.Accordingly, the crystalline forms of the compound of Formula I providedherein help satisfy the ongoing need for the development of NK-1antagonists for the treatment of serious diseases and disorders.

SUMMARY OF THE INVENTION

The present invention provides a crystalline form of the compound ofFormula I:

which is any one of Forms I, II, and III described herein.

The present invention further provides a crystalline form of thecompound of Formula I which is which is non-solvated.

The present invention further provides a crystalline form of thecompound of Formula I which is a trifluoroethanol solvate.

The present invention further provides a crystalline form of thecompound of Formula I which is a formate salt.

The present invention further provides a composition comprising acrystalline form of the invention and at least one pharmaceuticallyacceptable carrier.

The present invention further provides a process for preparing acrystalline form of the invention.

The present invention further provides a method of treating a diseaseassociated with activity of NK-1 receptor in a patient, comprisingadministering to the patient a therapeutically effective amount of acrystalline form of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 shows an XRPD pattern for Form I.

FIG. 2 shows the results of a DSC experiment for Form I.

FIG. 3 shows the results of a TGA experiment for Form I.

FIG. 4 shows the results of a DVS experiment for Form I.

FIG. 5 shows a microphotograph of Form I.

FIG. 6 shows (A) an overlay of a measured XRPD pattern for Form I (red)and the theoretical pattern (blue, calculated based on the singlecrystal structure at cryogenic temperature); and (B) theoretical powderdiffraction peaks and their Miller indices (hkl) as calculated from thecrystal structure.

FIG. 7 shows a ball and stick representation of the crystal structure ofForm I.

FIG. 8 shows crystal packing in the crystal of Form I. Thecrystallographic cell unit is shown in red.

FIG. 9 shows an IR spectrum for Form I.

FIG. 10 shows an NMR spectrum for Form I.

FIG. 11 shows an XRPD pattern for amorphous Formula I.

FIG. 12 shows the results of a DSC experiment for amorphous Formula I.

FIG. 13 shows the results of a TGA experiment for amorphous Formula I.

FIG. 14 shows the results of a DVS experiment for amorphous Formula I.

FIG. 15 shows an IR spectrum for amorphous Formula I.

FIG. 16 shows an NMR spectrum for amorphous Formula I.

FIG. 17 shows an XRPD pattern for Form II.

FIG. 18 shows an FT-Raman spectrum for Form II.

FIG. 19 shows the results of a TGA experiment for Form II.

FIG. 20 shows an XRPD pattern for Form III.

FIG. 21 shows an FT-Raman spectrum for Form III.

FIG. 22 shows the results of a TGA experiment for Form III.

FIG. 23 shows an XRPD pattern for Form I.

FIG. 24 shows an FT-Raman spectrum for Form I.

FIG. 25 shows an FT-IR spectrum for Form I.

FIG. 26 shows an XRPD comparison between a Form I reference sample(black line) and a ground sample (blue line).

FIG. 27 shows an XRPD comparison between a Form I reference sample(black line) and a kneaded sample (red line).

FIG. 28 shows an XRPD pattern for a micronized sample of Form I.

DETAILED DESCRIPTION

The present invention relates to, inter alia, crystalline forms of theNK-1 receptor antagonist2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidehaving Formula I:

which are useful, for example, in the preparation of solid dosage formsof the above compound for the treatment of various diseases, includingcancer.

Typically, different crystalline forms of the same substance havedifferent bulk properties related to, for example, hygroscopicity,solubility, stability, and the like. Forms with high melting pointsoften have good thermodynamic stability which is advantageous inprolonging shelf-life of drug formulations containing the solid form.Forms with lower melting points often are less thermodynamically stable,but are advantageous in that they have increased water solubility,translating to increased drug bioavailability. Forms that are weaklyhygroscopic are desirable for their stability to heat and humidity andare resistant to degradation during long storage. Anhydrous forms areoften desirable because they can be consistently made without concernfor variation in weight or composition due to varying solvent or watercontent. On the other hand, hydrated or solvated forms can beadvantageous in that they are less likely to be hygroscopic and may showimproved stability to humidity under storage conditions.

As used herein, “crystalline form” is meant to refer to a certainlattice configuration of a crystalline substance. Different crystallineforms of the same substance typically have different crystallinelattices (e.g., unit cells) which are attributed to different physicalproperties that are characteristic of each of the crystalline forms. Insome instances, different lattice configurations have different water orsolvent content. The different crystalline lattices can be identified bysolid state characterization methods such as by X-Ray Powder Diffraction(XRPD). Other characterization methods such as differential scanningcalorimetry (DSC), thermogravimetric analysis (TGA), dynamic vaporsorption (DVS), solid state NMR, and the like further help identify thecrystalline form as well as help determine stability and solvent/watercontent.

Crystalline forms of a substance include both solvated (e.g., hydrated)and non-solvated (e.g., anhydrous) forms. A hydrated form is acrystalline form that includes water in the crystalline lattice.Hydrated forms can be stoichiometric hydrates, where the water ispresent in the lattice in a certain water/molecule ratio such as forhemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also benon-stoichimetric, where the water content is variable and dependent onexternal conditions such as humidity.

Crystalline forms are most commonly characterized by XRPD. An XRPDpattern of reflections (peaks) is typically considered a fingerprint ofa particular crystalline form. It is well known that the relativeintensities of the XRPD peaks can widely vary depending on, inter alia,the sample preparation technique, crystal size distribution, filters,the sample mounting procedure, and the particular instrument employed.In some instances, new peaks may be observed or existing peaks maydisappear depending on the type of instrument or the settings (forexample, whether a Ni filter is used or not). As used herein, the term“peak” refers to a reflection having a relative height/intensity of atleast about 4% of the maximum peak height/intensity. Moreover,instrument variation and other factors can affect the 20 values. Thus,peak assignments, such as those reported herein, can vary by plus orminus about 0.2° (20), and the term “substantially” as used in thecontext of XRPD herein is meant to encompass the above-mentionedvariations.

In the same way, temperature readings in connection with DSC, TGA, orother thermal experiments can vary about ±4° C. depending on theinstrument, particular settings, sample preparation, etc. For example,with DSC it is known that the temperatures observed will depend on therate of the temperature change as well as the sample preparationtechnique and the particular instrument employed. Thus, the valuesreported herein related to DSC thermograms can vary, as indicated above,by ±4° C. Accordingly, a crystalline form reported herein having a DSCthermogram “substantially” as shown in any of the Figures is understoodto accommodate such variation.

The compound of Formula I can be isolated in numerous crystalline forms,including crystalline forms which are anhydrous, hydrated, non-solvated,or solvated. Example hydrates include hemihydrates, monohydrates,dihydrates, and the like. In some embodiments, the crystalline form ofthe compound of Formula I is anhydrous and non-solvated. By “anhydrous”is meant that the crystalline form of the compound of Formula I containsessentially no bound water in the crystal lattice structure, i.e., thecompound does not form a crystalline hydrate.

The compound of Formula I can also be isolated as a clathrate such thatthe stoichiometry of water to the compound of Formula I in thecrystalline lattice can vary without impacting the crystalline structureof the molecule. The degree of hydration (i.e. stoichiometric ratio ofwater to compound of Formula I) can range from greater than zero to asmuch as 3 without changing the crystalline form of the molecule. In someembodiments, the compound of Formula I has a degree of hydration of from0.5 to 2.5. In other embodiments, the crystalline form of the compoundof Formula I has a degree of hydration of from 1.0 to 2.0. Moreover, inany of these embodiments, the crystalline clathrate can further includean organic volatile impurity without impacting the crystalline structureof the molecule, such as methanol, ethanol, or isopropanol.

The compound of Formula I can also be isolated in crystalline saltforms. Crystalline salt forms of the invention can be prepared by anysuitable method for the preparation of acid addition salts. For example,the free base of the compound of Formula I can be combined with thedesired acid in a solvent or in a melt. Alternatively, an acid additionsalt of Formula I can be converted to a different acid addition salt byanion exchange. Crystalline salts of the invention which are prepared ina solvent system can be isolated by precipitation from the solvent.Precipitation and/or crystallization can be induced, for example, byevaporation, reduction of temperature, addition of anti-solvent, orcombinations thereof.

In some embodiments, the crystalline forms of the invention aresubstantially isolated. By “substantially isolated” is meant that aparticular crystalline form of the compound of Formula I is at leastpartially isolated from impurities. For example, in some embodiments, acrystalline form of the invention comprises less than about 50%, lessthan about 40%, less than about 30%, less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, less than about2.5%, less than about 1%, or less than about 0.5% of impurities.Impurities generally include anything that is not the substantiallyisolated crystalline form including, for example, other crystallineforms and other substances.

In some embodiments, a crystalline form of the compound of Formula I issubstantially free of other crystalline forms. The phrase “substantiallyfree of other crystalline forms” means that a particular crystallineform of the compound of Formula I comprises greater than about 80%,greater than about 90%, greater than about 95%, greater than about 98%,greater than about 99%, or greater than about 99.5% by weight of theparticular crystalline form.

In some embodiments, particularly the Crystalline Form I embodiments,the compound is present as a micronized compound. It has surprisinglybeen discovered that netupitant free base is well-absorbed when presentas Crystalline Form I, even superior to certain salts, and that thisabsorption can be improved even further by micronizing the compound. Inone embodiment, at least 90% of the particles are greater than 0.01 or0.1 microns and less than 500, 100, 50 or 10 microns.

Crystalline Form I

In some embodiments, the crystalline form of the compound of Formula Iis Form I. Form I is an anhydrous and non-solvated crystalline form ofthe compound of Formula I. This crystalline form can be generallyprepared by combining the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of toluene and n-heptane and heating the resultingmixture.

In some embodiments, the process for preparing crystalline Form Icomprises:

combining the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of toluene and n-heptane;

heating the mixture resulting from the combining of the compound andsolution;

filtering the heated mixture;

cooling the filtered mixture to afford a crystalline solid; and

isolating the crystalline solid.

In some embodiments, the heating step is performed at refluxtemperature.

In some embodiments, the cooling step is performed at a temperature of−10° C.

In some embodiments, the cooling step is performed for one hour at −10°C.

Crystalline Form I can be identified by unique signatures with respectto, for example, X-ray powder diffraction (XRPD), differential scanningcalorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vaporsorption (DVS). In some embodiments, crystalline Form I is characterizedby an XRPD pattern substantially as shown in FIG. 1. Peaks from the XRPDpattern are listed in Table 1.

In some embodiments, crystalline Form I is characterized by an XRPDpattern substantially as shown in FIG. 23. Peaks from the XRPD patternare listed in Table 9.

In some embodiments, crystalline Form I is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 4.50±0.2°. In someembodiments, crystalline Form I has an XRP pattern comprising thefollowing peaks, in terms of 2θ: 4.5°±0.2°; 8.4°±0.2°; 11.50° 0.2°;13.1°±0.2°; 13.9°±0.2°; 14.8°±0.2°; 16.7°±0.2°; 17.4°±0.2°; 17.7°±0.2°;19.5°±0.2°; 21.2°±0.2°; 21.6°±0.2°; 21.8°±0.2°. In some embodiments,crystalline Form I has an XRPD pattern comprising 2, or more, 3 or more,or 4 or more of the following peaks, in terms of 2θ: 4.50±0.2°;8.4°±0.2°; 11.50±0.2°; 13.10±0.2°; 13.90±0.2°; 14.80±0.2°; 16.70±0.2°;17.4°±0.2°; 17.7°±0.2°; 19.5°±0.2°; 21.2°±0.2°; 21.6°±0.2°; 21.8°±0.2°.

In some embodiments, Form I is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 160.3° C. Insome embodiments, crystalline Form I has a DSC thermogram substantiallyas shown in FIG. 2.

In some embodiments, crystalline Form I has a TGA trace substantially asshown in FIG. 3.

In some embodiments, crystalline Form I has a DVS trace substantially asshown in FIG. 4.

In some embodiments, crystalline Form I has an IR spectrum substantiallyas shown in FIG. 9.

In some embodiments, crystalline Form I has an NMR spectrumsubstantially as shown in FIG. 10.

In some embodiments, crystalline Form I has an FT-IR spectrumsubstantially as shown in FIG. 25

In some embodiments, crystalline Form I has an FT-Raman tracesubstantially as shown in FIG. 24.

Crystalline Form II

In some embodiments, the crystalline form of the compound of Formula Iis Form II. Form II is a crystalline trifluoroethanol solvate of thecompound of Formula I. This crystalline form can be generally preparedby combining the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of trifluoroethanol and water and heating the resultingmixture.

In some embodiments, the process for preparing crystalline Form IIcomprises:

combining the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of trifluoroethanol and water;

heating the mixture resulting from the combining of the compound andsolution;

filtering the heated mixture;

cooling the filtered mixture to afford a crystalline solid; and

isolating the crystalline solid.

In some embodiments, the heating step is performed at a temperature of70° C.

In some embodiments, the cooling step is performed at a temperature of3° C.

Crystalline Form II can be identified by unique signatures with respectto, for example, XRPD, DSC, TGA, and DVS. In some embodiments,crystalline Form II is characterized by an XRPD pattern substantially asshown in FIG. 17. Peaks from the XRPD pattern are listed in Table 7.

In some embodiments, crystalline Form II is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 4.00±0.2°. In someembodiments, crystalline Form II has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 4.0°±0.2°; 14.7°±0.2°; 15.5°±0.2°;16.6°±0.2°; 17.0°±0.2°; 17.4°±0.2°; 18.2°±0.2°; 19.9°±0.2°; 20.4°±0.2°;20.8°±0.2°; 21.2°±0.2°; 21.7°±0.2°; and 23.9°±0.2°. In some embodiments,crystalline Form II has an XRPD pattern comprising 2, or more, 3 ormore, or 4 or more of the following peaks, in terms of 2θ: 4.0°±0.2°;14.7°±0.2°; 15.5°±0.2°; 16.6°±0.2°; 17.0°±0.2°; 17.4°±0.2°; 18.2°±0.2°;19.9°±0.2°; 20.4°±0.2°; 20.8°±0.2°; 21.2°±0.2°; 21.7°±0.2; and23.9°±0.2°. In some embodiments, crystalline Form II has an XRPD patterncomprising the following peaks, in terms of 2θ: 4.0°±0.2°; 15.5°±0.2°;17.0°±0.2°; 18.2°±0.2°; 19.9±0.2°; 20.4±0.2; and 23.9°±0.2°.

In some embodiments, crystalline Form II has an FT-Raman tracesubstantially as shown in FIG. 18.

In some embodiments, crystalline Form II has a TGA trace substantiallyas shown in FIG. 19.

Crystalline Form III

In some embodiments, the crystalline form of the compound of Formula Iis Form III. Form III is a crystalline formate salt of the compound ofFormula I. This crystalline form can be generally prepared by combiningthe compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of formic acid and water.

In some embodiments, the process for preparing crystalline Form IIcomprises: combining the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidewith a solution of formic acid and water;

heating the mixture resulting from the combining of the compound andsolution;

filtering the heated mixture;

cooling the filtered mixture to afford a crystalline solid; and

isolating the crystalline solid.

In some embodiments, the heating step is performed at a temperature of23° C.

In some embodiments, the cooling step is performed at a temperature of4° C.

Crystalline Form III can be identified by unique signatures with respectto, for example, XRPD, DSC, TGA, and DVS. In some embodiments,crystalline Form III is characterized by an XRPD pattern substantiallyas shown in FIG. 20. Peaks from the XRPD pattern are listed in Table 8.

In some embodiments, crystalline Form III is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 8.00±0.2°. In someembodiments, crystalline Form III has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 4.0°±0.2°; 8.0°±0.2°; 10.0°±0.2°;12.0°±0.2°; 15.3°±0.2°; 16.0°±0.2°; 16.7°±0.2°; 18.4°±0.2°; 21.9±0.2°;22.10°±0.2°; 23.3°±0.2°; 23.4°±0.2°; 23.6°±0.2°; and 24.10±0.2°. In someembodiments, crystalline Form III has an XRPD pattern comprising 2, ormore, 3 or more, or 4 or more of the following peaks, in terms of 2θ:4.0°±0.2°; 8.0°±0.2°; 10.0°±0.2°; 12.0°±0.2°; 15.3°±0.2°; 16.0°±0.2°;16.7°±0.2°; 18.4°±0.2°; 21.9°±0.2°; 22.1°±0.2°; 23.3°±0.2°; 23.4°±0.2°;23.6°±0.2°; and 24.2°±0.2°. In some embodiments, crystalline Form IIIhas an XRPD pattern comprising the following peaks, in terms of 2θ:8.0°±0.2°; 10.0°±0.2°; 12.0°±0.2°; 16.0°±0.2°; 18.4°±0.2° and23.4°±0.2°.

In some embodiments, crystalline Form III has an FT-Raman tracesubstantially as shown in FIG. 21.

In some embodiments, crystalline Form III has a TGA trace substantiallyas shown in FIG. 22.

Methods

The crystalline forms of the invention are NK-1 receptor antagonistsparticularly useful for treating depression and pain, particularlydepression and pain resulting from inflammatory conditions (such asmigraine, rheumatoid arthritis, asthma, and inflammatory bowel disease)or disorders of the central nervous system (CNS) (such as Parkinson'sdisease or Alzheimer's disease). The crystalline forms of Formula I arefurther useful for the treatment of motion sickness and emesis.

The central and peripheral actions of the mammalian tachykinin substanceP have been associated with numerous inflammatory conditions includingmigraine, rheumatoid arthritis, asthma, and inflammatory bowel diseaseas well as mediation of the emetic reflex and the modulation of centralnervous system (CNS) disorders such as Parkinson's disease (Neurosci.Res., 1996, 7,187-214), anxiety (Can. J. Phys., 1997, 75, 612-621) anddepression (Science, 1998,281, 1640-1645). Evidence for the usefulnessof tachykinin receptor antagonists in pain, headache, especiallymigraine, Alzheimer's disease, multiple sclerosis, attenuation ofmorphine withdrawal, cardiovascular changes, oedema, such as oedemacaused by thermal injury, chronic inflammatory diseases such asrheumatoid arthritis, asthma/bronchial hyperreactivity and otherrespiratory diseases including allergic rhinitis, inflammatory diseasesof the gut including ulcerative colitis and Crohn's disease, ocularinjury and ocular inflammatory diseases is well established (“TachykininReceptor and Tachykinin Receptor Antagonists”, J. Auton. Pharmacol.,13,23-93, 1993). NK-1 receptor antagonists, in particular, are beingdeveloped for the treatment of a number of physiological disordersassociated with an excess or imbalance of tachykinin, in particularsubstance P. Examples of conditions in which substance P has beenimplicated include disorders of the central nervous system such asanxiety, depression and psychosis (WO 95/16679, WO 95/18124 and WO95/23798).

NK-1 receptor antagonists are further useful for the treatment of motionsickness and for treatment induced vomiting. The New England Journal ofMedicine, Vol. 340, No. 3 190-195, 1999 has been described the reductionof cisplatin-induced emesis by a selective neurokinin-1-receptorantagonist. U.S. Pat. No. 5,972,938 describes a method for treating apsychoimmunologic or a psychosomatic disorder by administration of atachykinin receptor, such as NK-1 receptor antagonist. Furthermore, thecrystalline forms of this invention are useful as agents againstheadache, anxiety, multiple sclerosis, attenuation of morphinewithdrawal, cardiovascular changes, oedema, such as oedema caused bythermal injury, chronic inflammatory diseases such as rheumatoidarthritis, asthma/bronchial hyperreactivity and other respiratorydiseases including allergic rhinitis, inflammatory diseases of the gutincluding ulcerative colitis and Crohn's disease, ocular injury andocular inflammatory diseases.

Some indications in accordance with the present invention are thosewhich include disorders of the central nervous system, for exampleindications for the treatment or prevention of certain depressivedisorders, anxiety or emesis by the administration of NK-1 receptorantagonists. A major depressive episode has been defined as being aperiod of at least two weeks during which, for most of the day andnearly every day, there is either depressed mood or the loss of interestor pleasure in all, or nearly all activities.

Further examples of NK-1-associated diseases include induced vomitingand nausea, including chemotherapy-induced nausea and vomiting (CINV)which is a common side effect of many cancer treatments. Furtherexamples of NK-1-associated diseases include overactive bladder disorder(OAB or urinary incontinence), which, in some cases, results fromsudden, involuntary contraction of the muscle in the wall of the urinarybladder.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the crystalline forms of the inventioncan be administered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routesdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (includingophthalmic and to mucous membranes including intranasal, vaginal andrectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or can be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration can include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the crystalline form of the invention incombination with one or more pharmaceutically acceptable carriers(excipients). In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active crystalline form, soft andhard gelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active crystalline form can be milled toprovide the appropriate particle size prior to combining with the otheringredients. If the active crystalline form is substantially insoluble,it can be milled to a particle size of less than 200 mesh. If the activecrystalline form is substantially water soluble, the particle size canbe adjusted by milling to provide a substantially uniform distributionin the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the crystalline forms or compositions of theinvention contain from about 5 to about 500 mg of the active ingredient.One having ordinary skill in the art will appreciate that this embodiescrystalline forms or compositions containing from about 50 to about 100,from about 100 to about 150, from about 150 to about 200, from about 200to about 250, from about 250 to about 300, from about 300 to about 350,from about 350 to about 400, from about 400 to about 450, or from about450 to about 500 mg of the active ingredient.

In some embodiments, the crystalline forms or compositions of theinvention contain from about 500 to about 1000 mg of the activeingredient. One having ordinary skill in the art will appreciate thatthis embodies crystalline forms or compositions containing from about500 to about 550, from about 550 to about 600, from about 600 to about650, from about 650 to about 700, from about 700 to about 750, fromabout 750 to about 800, from about 800 to about 850, from about 850 toabout 900, from about 900 to about 950, or from about 950 to about 1000mg of the active ingredient.

The active crystalline form can be effective over a wide dosage rangeand is generally administered in a pharmaceutically effective amount. Itwill be understood, however, that the amount of the crystalline formactually administered will usually be determined by a physician,according to the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual crystalline formadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acrystalline form of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the crystalline forms and compositions of thepresent invention can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of crystalline form or composition administered to a patientwill vary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the crystalline formpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the crystalline form of the present inventioncan vary according to, for example, the particular use for which thetreatment is made, the manner of administration of the crystalline form,the health and condition of the patient, and the judgment of theprescribing physician. The proportion or concentration of a crystallineform of the invention in a pharmaceutical composition can vary dependingupon a number of factors including dosage, chemical characteristics(e.g., hydrophobicity), and the route of administration. For example,the crystalline forms of the invention can be provided in an aqueousphysiological buffer solution containing about 0.1 to about 10% w/v ofthe crystalline form for parenteral administration. Some typical doseranges are from about 1 μg/kg to about 1 g/kg of body weight per day. Insome embodiments, the dose range is from about 0.01 mg/kg to about 100mg/kg of body weight per day. The dosage is likely to depend on suchvariables as the type and extent of progression of the disease ordisorder, the overall health status of the particular patient, therelative biological efficacy of the crystalline form selected,formulation of the excipient, and its route of administration. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems.

The crystalline forms of the invention can also be formulated incombination with one or more additional active ingredients which caninclude any pharmaceutical agent such as antibodies, immunesuppressants, anti-inflammatory agents, drugs used for the treatment ofrheumatoid arthritis, disorders of the central nervous system and thelike.

Labeled Compound and Assay Methods Another aspect of the presentinvention relates to labeled crystalline forms of the invention(radio-labeled, fluorescent-labeled, etc.) that would be useful not onlyin imaging techniques but also in assays, both in vitro and in vivo, forlocalizing and quantifying NK-1 in tissue samples, including human, andfor identifying NK-1 ligands by inhibition binding of a binding of alabeled compound. Accordingly, the present invention includes NK-1receptor assays that contain such labeled compounds.

The present invention further includes isotopically-labeled crystallineforms of Formula I. An “isotopically” or “radio-labeled” crystallineform is a crystalline form of the invention where one or more atoms arereplaced or substituted by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature(i.e., naturally occurring). Suitable radionuclides that may beincorporated in compounds of the present invention include but are notlimited to ²H (also written as D for deuterium), ³H (also written as Tfor tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl,⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide thatis incorporated in the instant radio-labeled crystalline form willdepend on the specific application of that radio-labeled crystallineform. For example, for in vitro NK-1 receptor labeling and competitionassays, crystalline forms that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I,³⁵S or will generally be most useful. For radio-imaging applications¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally bemost useful.

It is understood that a “radio-labeled” or “labeled crystalline form” isa crystalline form that has incorporated at least one radionuclide. Insome embodiments the radionuclide is selected from the group consistingof ³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br. Synthetic methods for incorporatingradio-isotopes into organic compounds are applicable to crystallineforms of the invention and are well known in the art.

A radio-labeled crystalline form of the invention can be used in ascreening assay to identify/evaluate compounds. In general terms, anewly synthesized or identified compound (i.e., test compound) can beevaluated for its ability to reduce binding of the radio-labeledcompound of the invention to the NK-1 receptor. Accordingly, the abilityof a test compound to compete with the radio-labeled compound forbinding to the NK-1 receptor directly correlates to its bindingaffinity.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of NK-1-associated diseaseswhich include one or more containers containing a pharmaceuticalcomposition comprising a therapeutically effective amount of acrystalline form of Formula I. Such kits can further include, ifdesired, one or more of various conventional pharmaceutical kitcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES

In the below examples, analytical grade solvents purchased from Fluka,ABCR or Merck were used unless otherwise stated.

X-Ray Powder Diffraction (XRPD) patterns were recorded in transmissiongeometry on a STOE STADI P diffractometer with CuKα radiation (1.54 Å)and a position sensitive detector. The samples (approximately 50 mg)were prepared between thin polymer films and analyzed without furtherprocessing (e.g., grinding or sieving) of the substance unless otherwiseindicated.

XRPD patterns were alternatively recorded on a Bruker D8 diffractometerwith CuKα radiation (40 kV/40 mA) and a LynxEye detector. Alternatively,XRPD patterns were recorded on a X'Pert PRO diffractometer using aPW3065 Goniometer.

Differential Scanning Calorimetry (DSC) was carried out on aMettler-Toledo differential scanning calorimeter DSC820 with a FRS05sensor. System suitability tests and calibrators were carried outaccording to the internal standard operation procedure. The generalexperimental conditions were 30° C. to a maximum temperature of either180 or 220° C. at 5 K/min or 10K/min nitrogen gas flow at 100 mL/min,using an aluminum sample pan.

Thermogravimetric analysis (TGA) was carried out on a Mettler-Toledothermogravimetric analyzer (TGA850/SDTA) with the following conditions:Ramp at 5 K/min to 220° C.; nitrogen gas at 50 mL/min sample purge flow;aluminum sample pan.

TGA measurements were alternatively conducted on a NetzschThermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector22 using the following conditions: Ramp at 10° C./min under nitrogen;aluminum sample pan equipped with pinholes.

Infrared (IR) spectra were recorded as film of a suspension in Nujolconsisting of approximately 15 mg of sample and approximately 15 mg ofNujol between two sodium chloride plates, with a FTIR spectrometerNicolet 20SXB in transmittance (resolution 2 cm⁻¹, 200 or more co-addedscans, MTC detector). Alternatively, the spectra were recorded withoutpreparation in attenuated total reflection mode (ATR) with an FTIRspectrometer equipped with an IR-Microscope (Nic-Plan Nicolet)(resolution 2 cm⁻¹, 200 or more co-added scans, MTC detector).

Single crystal structure analysis was collected on a STOE Image PlateDiffraction System (STOE, Darmstadt) with Mo-radiation (0.71 Å) and dataprocessed with STOE IPDS-software. The crystal structure was solved andrefined with ShelXTL (Bruker AXS, Karlsruhe).

Moisture Adsorption/Desorption data was collected on a DVS-1 (SMSSurface Measurements Systems) moisture balance system. Thesorption/desorption isotherms were measured stepwise in a range of 0% RHto 90% RH at 25° C. A weight change of <0.002 mg/min was chosen as thecriterion to switch to the next level of relative humidity (with amaximum equilibration time of 6 hours if the weight criterion was notmet). The data were corrected for the initial moisture content of thesamples so that the weight after drying the sample at 0% relativehumidity was taken as the zero point. The hygroscopicity of a givensubstance was characterized, in accordance with the EuropeanPhamacopoeia (Technical Guide 1999), by the increase in mass when therelative humidity is raised from 0% RH to 90% RH, as defined below(where weight increase=x):

-   -   Non hygroscopic: x<0.2%    -   Slightly hygroscopic: 0.2%≤x<2.0%    -   Hygroscopic: 2.0%≤x<15%    -   Very hygroscopic: x≥15.0%    -   Deliquescent: Sufficient liquid as adsorbed to form a liquid

FT-Raman Spectra were recorded on a Bruker RFS 100 FT-Raman system witha near infrared Nd:YAG laser operating at 1064 nm and a liquidnitrogen-cooled germanium detector. For each sample, 64 scans with aresolution of 2 cm⁻¹ were accumulated. 300 mW laser power was used. TheFT-Raman data are shown in the region between 3500 to 100 cm⁻¹.

NMR spectra were recorded using a Bruker DPX300 spectrometer.

Example 1 Preparation and Characterization of Form I

Crystalline Form I was prepared by combining 179 g of the compound ofFormula I with toluene (179 g) and n-heptane (585 g) and the solutionwas heated to reflux temperature and filtered to afford a clearsolution. The solution was then cooled to −10° C. at 10 K/h. After agingof the suspension for 1 h at this temperature, crystals were isolatedand dried at 80° C./10 mbar overnight.

Form I was confirmed as a crystalline solid according to XRPD analysis.The XRPD pattern of Form I is shown in FIG. 1 and the peak data is givenbelow in Table 1.

TABLE 1 XRPD Peak Data for Form I. Peak No. 2-Theta 1 4.5 2 8.4 3 11.5 413.1 5 13.9 6 14.8 7 16.7 8 17.4 9 17.7 10 19.5 11 21.2 12 21.6 13 21.8

DSC analysis of Form I revealed one melting endotherm peak with an onsettemperature of 159.5° C. (varying between 158.3-160.6° C.) and a maximumat 160.3° C. (varying between 159.9-162.6). The DSC thermogram isprovided in FIG. 2.

TGA analysis of Form I revealed a <0.1% weight loss up to 140° C. Aftermelting, a continuous weight loss above 160° C. indicated the startingdecomposition of the compound. The TGA thermogram is provided in FIG. 3.

Moisture adsorption/desorption of Form I was analyzed by DVS. Resultsfrom two DVS cycles are shown in FIG. 4. The data indicates that, duringthe drying step and the first adsorption segment, Form I exhibits aweight gain of about 0.3% which is likely due to electrostatic charges.A 0.1% weight loss was observed in the second cycle. The shapes of theisotherms indicated that Form I is non-hygroscopic and unsolvated.

Single crystal parameters for Form I are shown in Table 2. Amicrophotograph showing Form I crystals is provided in FIG. 5. Thetheoretical powder pattern calculated based on the crystal structurematches the measured powder pattern well and allows the assignment ofMiller indices (hkl) to some reflections (FIG. 6). Small differences inpeak positions between theoretical and measured patterns are believed tobe due to the changes in dimension of the crystal unit cell whenchanging the temperature from room temperature to 150 K and make theassignment of indices of reflections at higher 20 values difficult. Anoverlay of the measured XRPD pattern of Form I and the theoreticalpattern of Form I based on the single crystal structure at cryogenictemperature is shown in FIG. 6.

TABLE 2 Single Crystal Parameters of Form I. Parameter MeasurementCrystal system Triclinic Space group P-1 Crystal habit Needle Like Unitcell dimensions a = 12.173 (Å) b = 12.556 c = 19.247 (°) α = 89.97   β =87.38  γ = 83.34  Temperature 150 (K) Cell volume 2918 (Å³) Molecules incell unit 4 Density (calculated) 1.317 (g/cm³)

Crystals of Form I were found to contain two molecules per asymmetricunit. No solvent molecules are present in the crystal lattice. The twomolecules per asymmetric unit both assume a similar conformation asshown in FIG. 7. The crystal packing contacts are mainly hydrophobic andseveral interactions between fluorine atoms in the crystal latticepacking are visible as shown in FIG. 8.

IR analysis of Form 1 revealed specific bands at about 1647, 1610, 1598,1534, 1500, 1403, 1375, 1367, 1339, 1330, 1278, 1233, 1187, 1171, 1149,1081, 1005, 902, 895, 845, 803, 769, 711, and about 706 cm⁻¹. The IRspectrum is shown in FIG. 9.

The NMR spectrum of Form I is shown in FIG. 10.

Selected physicochemical data of Form I is summarized below in Table 3.

TABLE 3 Physicochemical data of Form I. Parameter Measurement Meltingtemperature by DSC T_(onset) = 159.5 (158.3-160.6) (° C.)T_(extrapol. peak) = 160.3 (159.9-162.6) Heat of fusion 36.7 (34.8-37.4)(kJ/mol) Entropy of fusion 84.6 J/(mol*K) Weight loss between 25° C. and<0.1 (<0.1-0.2) 140° C. (%) Density Calculated = 1.317 (g/cm³) Measured= 1.33 Hygroscopicity (weight change 0 0.1 (non-hygroscopic) to 90%-RHclassification) (%) FTIR spectrum 1647, 1610, 1598, 1534, 1500, 1403,(cm⁻¹) 1375, 1367, 1339, 1330, 1278, 1233, 1187, 1171, 1149, 1081, 1005,902, 895, 845, 803, 769, 711, and about 706 XRPD peaks 4.5, 8.4, 11.5,13.1, 13.9, 14.8, 16.7, (2θ) 17.4, 17.7, 19.5, 21.2, 21.6, 21.8

Samples of Form I were equilibrated in various solvents at 25° C. or 60°C. to test the solubility of Form I in the solvents. Solubility wasdetermined gravimetrically. A weighted sample of Form I was suspended ina defined amount of solvent. After equilibration and solvent-liquidseparation, the weight of the saturated liquid was determined. Thesolvent was then evaporated, the solid residue dried to dryness andweighed. Results of the solubility experiments are shown in Tables 4 and5. The solubility is reported as weight of solid substance dissolveddivided by the weight of the solution.

TABLE 4 Solubility of Form I at 25° C. Solvent Solubility (% m/m) Water<0.1 Ethanol >11.5 Methanol >11.9 2-Propanol N/A 1-Butanol >11.6Acetone >11.1 N,N-Dimethylformamide >9.9 Trifluoroethanol >7.5Tetrahydrofuran >9.9 Acetonitrile 9 Dioxane >9.0 Dichloroethane >7.0Ethyl Acetate N/A 2-Butanone >11.2 Toluene >10.8 Water/Acetonitrile 2:10.5 Water/Acetonitrile 1:2 6.2 Water/Dioxane 2:1 0.1 Water/Dioxane 1:24.9 Water/Ethanol 2:1 0.1 Water/Ethanol 1:2 3.1 Water/Methanol 2:1 0.2Water/Methanol 1:2 1.0 Water/Acetone 2:1 0.2 Water/Acetone 1:2 7.0Heptane 1.2 Cyclohexane 4.2 Isopropyl Acetate 22.2 Xylene 18.4

TABLE 5 Solubility of Form I at 60° C. Solvent Solubility (% m/m) Water<0.1 Ethanol 33.1 Methanol 48.4 2-Propanol 26.2 1-Butanol 29.9Acetone >53.3 N,N-Dimethylformamide 40.7 Trifluoroethanol >39.1Tetrahydrofuran >50.4 Acetonitrile 14.4 Dioxane >41.6 Dichloroethane34.7 Ethyl Acetate 45.9 2-Butanone >52.6 Toluene 48.8 Water/Acetonitrile2:1 <0.1 Water/Acetonitrile 1:2 6.4 Water/Dioxane 2:1 <0.1 Water/Dioxane1:2 21.1 Water/Ethanol 2:1 <0.1 Water/Ethanol 1:2 5.5 Water/Methanol 2:1<0.1 Water/Methanol 1:2 0.4 Water/Acetone 2:1 <0.1 Water/Acetone 1:211.7 Heptane 3.0 Cyclohexane 16.0 Isopropyl Acetate >35.0 Xylene >35.0

Example 2 Preparation and Characterization of Amorphous Form

The amorphous form of the compound of Formula I was prepared bydissolving Form I (5 g) in dioxane (50 mL) in an ultrasonic bath. Afterfiltration, the resulting clear solution was frozen (dry ice/acetonebath) and dried.

The amorphous nature of the material was confirmed by XRPD analysis. TheXRPD pattern of the amorphous form of the compound of Formula I shown inFIG. 11.

DSC analysis of the amorphous form of the compound of Formula I revealeda one melting endotherm peak with an onset temperature of 41.4° C. and amaximum at 46.4° C. In particular, upon heating the amorphous form ofthe compound of Formula I to 70° C., a glass transition was observedbetween about 50° C. and 65° C. The sample was cooled to 0° C. and thenreheated to yield a glass transition between about 40° C. and 60° C.with minimal relation enthalpy allowing a more accurate determination ofthe glass transition temperature (midpoint 46.4). Upon further heating,the sample crystallized in the temperature range of about 80° C. toabout 120° C. to yield Form I. The DSC thermogram of the amorphous formis provided in FIG. 12.

The TGA thermogram of the amorphous form of the compound of Formula I isprovided in FIG. 13.

Moisture adsorption/desorption of Form I was analyzed by DVS. Resultsfrom two DVS cycles are shown in FIG. 14. The data indicates that, theamorphous material adsorbs up to 1.5% w/w of moisture. Nocrystallization could be observed during the sorption isothermmeasurement.

The IR spectrum of the amorphous form of the compound of Formula Irevealed specific bands at about 1647, 1609, 1596, 1484, 1395, 1364,1275, 1232, 1184, 1171, 1127, 1079, 1005, 956, 894, 844, 766, 748, 731,and about 708 cm⁻¹. The IR spectrum is shown in FIG. 15.

The NMR spectrum of the amorphous form of the compound of Formula I isshown in FIG. 16.

Selected physicochemical data of the amorphous form of the compound ofFormula I is summarized below in Table 6.

TABLE 6 Physicochemical Data of Amorphous Form. Parameter MeasurementWeight loss between 25° C. and  0.2 140° C. (%) Glass transitiontemperature 46.4 (° C.) Hygroscopicity (weight change 0 1.5 (slightlyhygroscopic) to 90%-RH classification) (%) FTIR spectrum 1647, 1609,1596, 1484, 1395, 1364, (cm⁻¹) 1275, 1232, 1184, 1171, 1127, 1079, 1005,956, 894, 844, 766, 748, 731, 708

Example 3 Preparation and Characterization of Form II

Crystalline Form II was prepared by dissolving trifluoroethanol in waterat a 5:4 ratio at 70° C., and cooling at 3° C./hour. An emulsion wasinitially obtained, partial evaporation and additional oftrifluoroethanol/water at room temperature led to Form II. Form II wasfound to be a crystalline trifluoroethanol solvate.

Form II was confirmed as a crystalline solid according to XRPD analysis.The XRPD pattern of Form II is shown in FIG. 17 and the peak data isgiven below in Table 7. Form II was found to be unstable under ambientconditions and converts to Form I. FIG. 17 shows the XRPD pattern ofForm II (black) in comparison with Form I (blue). The red pattern wasrecorded after storing Form II for 1 h and 20 min under ambientconditions. A partial conversion to Form I was observed.

TABLE 7 XRPD Peak Data for Form II. 2-Theta H % 4.0 39.5 14.7 33.8 15.534 16.6 40.1 17.0 48.3 17.4 33.2 18.2 81.4 19.9 80.3 20.4 60.6 20.8 38.221.2 100 21.7 69.6 23.9 62.8

FT-Raman Spectroscopy analysis of Form II is provided in FIG. 18 withthe most pronounced Raman peaks labeled in the figure. The FT-Raman dataare shown in the region between 3500 and 100 cm⁻¹.

TGA analysis of Form II revealed a 41% weight loss up to 130° C. Theweight loss is attributed to water and trifluoroethanol (monohydrate:3%, monosolvate: 14.7%). The TGA thermogram of Form II is provided inFIG. 19.

Form II was found to convert to Form I at 80% relative humidity.Thermogravimetry coupled to Fourier Transform (TGA-FT) measurementstaken during the conversion show a loss of trifluoroethanol above 100°C. Form II was also found to convert to Form I upon suspensionequilibration in water.

Example 4 Preparation and Characterization of Form III

Crystalline Form III was prepared by cooling a solution of Form I in aformic acid/water mixture to 4° C.

Form III was found to be a formate salt.

Form III was confirmed as a crystalline solid according to XRPDanalysis. The XRPD pattern of Form III is shown in FIG. 20 and the peakdata is given below in Table 8.

The approximate solubility of Form III in water at rt is below 1 mg/mL.

TABLE 8 XRPD Peak Data for Form III. 2-Theta H % 4.0 58.6 8.0 70.9 10.032.7 12.0 67.9 15.3 71 16.0 100 16.7 37.7 18.4 57.8 21.9 43.5 22.1 6523.3 54.2 23.4 72.4 23.6 31.5 24.2 35.4

FT-Raman Spectroscopy analysis of Form III is provided in FIG. 21 withthe most pronounced Raman peaks labeled in the figure. The FT-Raman dataare shown in the region between 3500 and 100 cm⁻¹.

¹H and ¹³C-NMR spectroscopy of Form III were found to be consistent withthe formation of a monosalt.

TGA analysis of Form III revealed a 1.7% weight loss up to 115° C. Theweight loss is consistent for a non-stoichiometric hydrate or forsurface adsorbed water. Above 115° C. a mass loss of about 10% isobserved, which is attributable to formic acid, water and decomposition(theoretical mass loss for a monosalt: 7.4% formic acid). The TGAthermogram of Form III is provided in FIG. 22

Example 5 Supplemental Characterization of Form I

A micronized sample of Form I was characterized by Powder X-RayDiffraction (XRPD). The XRPD spectrum for Form I is provided in FIG. 23and the corresponding peak data is provided below in Table 9.

TABLE 9 XRPD Peak Data for Form I. 2-Theta H % 4.5 35.3 8.4 57.6 11.441.5 13.1 35.3 13.9 37.6 14.7 36.2 16.7 72.9 17.3 100 17.6 42.7 19.545.8 20.7 55.6 21.2 55 21.5 86 21.8 62 22.1 39.5 22.9 45.7 23.7 55.3

FT-Raman Spectroscopy analysis of Form I is provided in FIG. 24 with themost pronounced Raman peaks labeled in the figure. The FT-Raman data areshown in the region between 3500 and 100 cm⁻¹.

The pKa of Form I was calculated to be 6.1 and 7.9. PKa calculationswere conducted using an ACD/Labs device (Release 10; Product Version10).

Example 6 Supplemental Characterization of Form I

The compound of Formula I (Form I) was further characterized by theprocedures described below. A micronized batch of the compound ofFormula I (batch #27005937 from Helsinn Chemicals SA) was used as thestarting material in the characterization experiments unless otherwisenoted.

The micronized sample (Form I) was characterized by XRPD. The XRPDspectrum is provided in FIG. 28 and the associated peaks are shown belowin Table 10.

TABLE 10 XRPD Peak Data for Micronized Sample. 2-Theta Height (cts) H %4.6 1047.62 53.59 8.5 1954.88 100.00 10.6 90.86 4.65 11.6 1016.25 51.9913.2 658.90 33.71 14.0 686.68 35.13 14.8 553.25 28.30 15.5 340.72 17.4315.8 263.63 13.49 16.2 127.06 6.5 16.8 1191.76 60.96 17.5 1460.76 74.7217.8 773.60 39.57 18.4 245.75 12.57 19.1 137.2 7.02 19.6 580.31 29.6919.9 474.34 24.26 20.5 466.73 23.87 20.8 617.14 31.57 21.3 626.52 32.0521.7 1059.15 54.18 22.0 845.02 43.23 22.3 493.18 25.23 23.0 335.23 17.1523.9 481.59 24.64 24.3 211.54 10.82 24.8 148.87 7.62 25.7 97.43 4.9826.0 131.94 6.75 27.7 69.51 3.56 28.1 75.40 3.86 28.5 62.66 3.21 29.241.70 2.13 30.7 51.99 2.66 31.8 71.50 3.66 33.0 34.37 1.76 34.3 46.792.39 35.4 41.40 2.12 37.2 49.38 2.53 38.8 29.80 1.52

FT-IR analysis of Form I used in the experiments below revealed specificbands as shown in Table 11. The FT-IR spectrum of Form I is shown inFIG. 25.

TABLE 11 FT-IR Analysis of Form I. Wavenumber (cm⁻¹) Intensity 662.281.3 681.6 57.3 695.9 82.6 710.1 61.8 730.9 72.2 745.7 72.5 766.7 70.2802.8 85.3 834.5 80.1 844.1 75.9 857.0 84.8 870.9 85.4 894.0 65.2 901.165.1 924.2 86.5 956.9 75.8 1004.8 70.8 1049.6 82.1 1079.6 54.1 1100.163.8 1134.6 34.2 1170.5 51.3 1185.4 53.8 1231.7 66.2 1275.9 30.8 1330.385.6 1364.7 59.9 1401.5 80.0 1443.6 80.0 1471.9 77.7 1499.2 75.6 1533.291.7 1597.2 75.5 1609.5 75.4 1646.2 62.7 2793.9 91.2 2845.8 92.1 2931.992.4 2974.3 94.7

Grounded Form I

An untreated sample of Form I was ground by ball milling in a Retsch MM200 grinder for 10 min at a frequency of 30 Hz. The sample was thenanalyzed by XRPD to determine its diffraction pattern. The stability ofthe ground sample was determined by comparing the diffraction pattern ofthe ground sample with that of the reference sample. As shown in FIG.26, grinding caused loss of crystallinity of the sample. In FIG. 26, theXRPD pattern for the Form I reference sample is shown in black and thepattern for the ground sample is shown in blue.

Kneaded Form I

An untreated sample of Form I was ground by ball milling in a Retsch MM200 grinder for 10 min at a frequency of 30 Hz with a catalytic amountof water. The sample was then analyzed by XRPD to determine itsdiffraction pattern. The stability of the kneaded sample was determinedby comparing the diffraction pattern of the kneaded sample with that ofthe reference sample. As shown in FIG. 27, kneading caused increaseddegree of crystallinity with more defined peak separation. In FIG. 27,the XRPD pattern for the Form I reference sample is shown in black andthe XRPD pattern for the kneaded sample is shown in red.

Example 7

Pharmacokinetics of the Compound of Formula I after Intravenous and/orOral Administration of the Free Base and of Different Salts in Dogs

In Example 10, the pharmacokinetics of the compound of Formula I in dogsfollowing single intravenous and oral administration was evaluated andthe oral bioavailability of five different salts of the compound ofFormula I was compared. The compound of Formula I was administered todogs as described in Table 12. The doses are expressed in mg of freebase.

TABLE 12 Formula I Dosage Forms and Administration. ProtocolAdministration Formu- Sample Ref. Route lation Dose Hydrochloride Salt Ap.o Capsule 120 mg/dog Malate salt A p.o Capsule 120 mg/dog TartrateSalt A p.o Capsule 120 mg/dog Free Base A p.o Capsule 120 mg/dog FreeBase B p.o Capsule  70 mg/dog Tartrate B p.o Capsule  70 mg/dog MesylateB p.o Capsule  70 mg/dog

Test Animals

The experiments were performed in 15 male dogs (bodyweight 11-18 kg)from RCC Ltd., Biotechnology and Animal Breeding Division, Wolferstrasse4, CH-4414, Fullinsdorf, Switzerland. The animals were housed understandard laboratory conditions throughout the study period. They werefasted overnight before drug administration and had free access to waterand food during the experiment.

Sample Collection

For the pharmacokinetic studies, 1 mL blood samples were collected fromthe cephalic vein of the dogs via catheter at 15, 30 min and at 1, 2, 3,4, 6, 8, 24, 32, 48, 56, 72, 80, 96, and 100 (or 104) hours followingoral dosing. After intravenous administration, blood samples werecollected at 5, 15, 30 min and 1, 2, 3, 4, 6, 8, 24, 32, 48, 56, 75,102, 120, 128, 144, 152, 168, 176, 192, and 200 hours. Collection tubescontained EDTA/NaF as anticoagulant and stabilizer. Aftercentrifugation, plasma was removed and stored deep frozen atapproximately −20° C. until analysis using a specific LC-MS method.

Analytical Methods

Protocol A

Aliquots of 40 μL plasma were mixed with 50 μL buffer at pH 5, 50 μLinternal standard of Formula I (1 μg/mL in 1-chlorobutane) and 250 μLbutylacetate and then shaken for 10 min. After centrifugation, 200 μL ofthe supernatant were evaporated to dryness at 45° C. under a stream ofnitrogen. The residue was reconstituted with 200 μL acetonitrile/1%formic acid in water (30/70, v/v). Aliquots of 30 μL were injected ontothe analytical column (Waters, Symmetry C8, 2.1×150 mm, 5 μm).

Separation occurred by gradient elution using a solvent A (acetonitrile)and solvent B (1% formic acid in water). The flow rate was 0.3 mL/minand the gradient elution was:

Time Mobile Phase Mobile Phase (min) A (%) B (%) 0 30 70 5 60 40 9 60 4010 30 70The sample passed to the ion-spray interface of the single quadrupolemass spectrometer. Selected ion monitoring mode (SIM) was used for massspectrometric detection. Quantification was based on peak area ratiosand calibration curve established between 2 and 5000 ng/mL.

Protocol B

To a 100 μL sample aliquot were added 200 μL of a mixture ofacetonitrile/ethanol containing the deuterated internal standard, toprecipitate plasma proteins. After vortex mixing and centrifugation,aliquots of the supernatants were transferred to 96-deel-well plates andinjected for analysis (5 μL). Following enrichment and cleanup on thetrapping column, the analyte was eluted and separated by gradientelution on a 2.1*30 mm analytical column (XTerra MS C18). The effluentfrom the analytical column was passed to the turbo ion spray vie adivert valve.

Calibration standards were prepared in dog plasma. The calibration rangewas from 10-5000 ng/mL. In each analytical series, a set of calibrationstandards was worked up and run with the unknowns. Calibration was thenperformed by computing a weighted (1/X²) least-squares linear regressionline of the measured peak area ratios (Y) (analyte to internal standard)versus the spiked concentrations (X). The drug concentrations of theunknown samples were then calculated from this regression line(UNICHROM). The performance of the analytical procedure during sampleanalysis was monitored. With each analytical series, quality control(QC) samples in dog plasma, spiked with known amounts of the analyte,were run together with the study samples.

Not all samples were collected at the times specified in the protocols.This did not compromise the outcomes of the study, as the actualsampling times were recorded and were used for the pharmacokineticanalysis.

Data Processing

Data acquisition and integration was performed using software packagesSample Control and MacQuan from PE Sciex. MacQuan was additionally usedto generate an ASCII file, containing the relevant sample information ofthe actual analytical series to be used for the calculation of theregression line and of the drug concentrations of the unknowns. Thisfile was transferred to the VAX based software package Unichrom 1.5 forfurther concentration calculations. The calculated analytical resultswere then stored in the database Kinlims.

Kinetic Analysis

The pharmacokinetic parameters were estimated by non-compartmentalanalysis, using the pharmacokinetic evaluation program WinNOnlin [1].AUC(0-inf.) was calculated applying the linear trapezoidal rule andextrapolation to infinity using the apparent elimination rate constantλz and the calculated concentration at the last measurable time point.AUClast values were calculated by linear trapezoidal rule from time zeroto time of last measurable time point. Cmax, C(t), and Tmax weredetermined directly from the plasma concentration-time profiles. Theapparent terminal half-life (T_(1/2)) was derived from the equation:T_(1/2)=ln 2/λz. Means of the half-life were calculated by harmonicmeans. Plasma clearance, CL, was calculated as D/AUC(0-inf.). Volume ofdistribution, Vz, was calculated as CL/λz. The absolute bioavailabilitywas calculated from plasma concentration data as follows:

${F(\%)} = {\frac{{{AUC}\left( {0 - \infty} \right)}_{p.o.}}{{{AUC}\left( {0 - \infty} \right)}_{i.v}}*\frac{D_{i.v.}}{D_{p.o.}}*100}$

Possible small deviations of the reported mean values from thosecalculated from non-rounded pharmacokinetic parameters are due to therounding procedure of individual values.

Assay Performance

The performance of the LC-MS assay was assessed from the analysis ofquality control samples, which were measured alongside the unknownsamples.

For Protocol A, the average inter-assay precision was 6.3% in theconcentration range 2-5000 ng/mL plasma and the correspondinginter-assay inaccuracy averaged 11% for plasma. The quantification limitwas set to 4 ng/mL (20% below the lowest calibration point). This wasconsidered to be adequate to reach the objective of the study.

For Protocol B, the average inter-assay precision was 5.1% in theconcentration range of 10-5000 ng/mL plasma and the correspondinginter-assay inaccuracy averaged 4.4% for plasma. The quantificationlimit was set to 10 ng/mL. This was considered to be adequate to reachthe objective of the study.

The calculated pharmacokinetic parameters are compiled in Tables 13-14.

TABLE 13 Pharmacokinetics of Formula I Following Single OralAdministration of 120 mg Free Base and Salts to dogs. FormulationHydrochloride Malate Tartrate Free Base Dog No. 6461 5563 6464 6452 64826460 2375 5547 Dose* 8.6 8.7 7.8 8.0 10.3 10.1 8.0 7.9 (mg/kg) Cmax 1110733 1430 1090 1160 1970 614 757 (ng/mL) Tmax (h) 1.0 4.0 3.0 4.0 6.0 3.02.0 2.0 AUC (0-100 h) 25900 20900 40000 21600 26300 45500 11000 17400(ng · h/mL) AUC (0-Inf.) 36400 26900 58100 26300 28800 55800 12700 22600(ng · h/mL) T_(1/2) (h) 64.0 48.2 64.4 44.0 35.0 42.6 39.5 56.3 F (%)91.4 66.7 161 71.0 60.3 119 34.3 61.7 F** (%) — — 130 — — 115 — — *Doseexpressed in mg of free base **Calculated with mean AUC(0-102 h) of11800 ng · h/mL after i.v. application of 3 mg/kg Formula I (n = 3)

TABLE 14 Pharmacokinetics of Formula I Following Single OralAdministration of 70 mg Free Base and Salts to dogs. Formulation FreeBase Tartrate Mesylate Dog No. 6460 5455 6396 6452 6392 6393 Dose* 5.04.9 5.1 5.4 5.4 6.3 (mg/kg) Cmax 587 667 1120 358 320 287 (ng/mL) Tmax(h) 6 4 2 3 4 3 AUC (0-100 h) 16200 12800 23400 10700 6380 7550 (ng ·h/mL) AUC (0-Inf.) 19300 14500 31000 12300 7150 8100 (ng · h/mL) T_(1/2)(h) 40.4 35.7 53.1 34.8 41.2 26.7 F (%) 83.3 63.9 131 49.2 28.6 27.7 F**(%) — — 117 — — — *Dose expressed in mg of free base **Calculated withmean AUC(1-102 h) of 11800 ng · h/mL after i.v. application of 3 mg/kgFormula I (n = 3)

Oral Administration

The free base of the compound of Formula I and three different salts ofFormula I (hydrochloride, malate, and tartrate) were administered orallyin a gelatin capsule (120 mg/capsule calculated as free base) to twodogs per salt. In a further experiment, two different salts of thecompound of Formula I (tartrate and mesylate) and free base wereadministered orally in a gelatin capsule (70 mg/capsule calculated asfree base) to two dogs per salt.

After oral administration of the compound of Formula I as a free base totwo dogs (Protocol A) Cmax values of 614 and 757 ng/mL were achieved at2 hours following the administration. In these two animals the oralbioavailabilities were 40 and 56%, respectively. In a second experiment(Protocol B), after administration of lower doses of Formula I, Cmax of587 and 667 ng/mL were achieved between 4 and 6 hours after dosing. Theoral bioavailabilities were slightly higher than in the first experimentwith oral bioavailability values of 83 and 64% respectively. In the fouranimals, the apparent terminal half-lives ranged between 36 and 56hours. For these two experiments, two different batches of the compoundof Formula I were used. A difference in particle size might explain theobserved differences between both experiments. For the first batch,which was not milled, the particle size was estimated to be about 10 to20 μm, whereas in the second batch which was fine milled the measuredparticle size was 3-6 μm. In addition, these experiments were conductedin different animals (parallel groups) and the difference inbioavailability might also be attributed to inter-individualvariability.

After oral administration of the hydrochloride salt to two dogs, Cmaxvalues of 1110 and 733 ng/mL were achieved at 1 and 4 hours afterdosing, respectively. The systemic bioavailabilites were at 91 and 67%in both animals. The apparent terminal half-lives (64 and 44 hours,respectively) were similar to those observed in other animals.

Oral administration of the malate salt to two dogs showed Cmax values of1430 and 1090 ng/mL, which were achieved 3 to 4 hours following dosing.The oral bioavailability values were 161 and 70%, respectively. The oneanimal with the bioavailability value of 161% showed a flat plasmaconcentration-time profile, with a terminal half-life of 64 hours.Therefore, the oral bioavailability in this animal might beoverestimated by using extrapolated areas under the curve values. Withtruncated areas under the curves (AUC0-1-104 h), the oralbioavailability of Formula I in this animal was still 130%. The reasonfor this high value remained unclear.

After oral application of the tartrate salt to four dogs, Cmax valueswere 1160 and 1970 ng/mL (120 mg dose) and 1120 and 358 ng/mL (70 ngdose). Peak concentrations were achieved between 2 and 6 hours afterdosing. The apparent terminal half-lives ranged between 35 and 53 hoursand the systemic bioavailabilities ranged between 49 and 131%.

After oral administration of the mesylate salt to 2 dogs, Cmax valueswere lower than with the base and the different other salts tested. Theywere 320 and 287 ng/mL and were achieved at 4 and 3 hours after dosing,respectively. The systemic bioavailabilities were 29 and 28%,respectively. The apparent terminal half-lives were 41 hours and 27hours.

During the course of the experiments, no overt pharmacological ortoxicological signs were observed in dogs.

Conclusions

The results indicated an oral bioavailability of Formula I ranging from34 to 83% following administration of the free base in gelatin capsuleform. The results also indicate an oral bioavailability of Formula Iranging from 28 to 160% following oral administration of Formula I inthe form of different salts (hydrochloride, malate, tartrate, mesylate)in gelatin capsule form, the lowest bioavailability being observed withthe mesylate salt (28%). Based on pharmacokinetic as well as ongalenical considerations, the free base was considered as suitable forfurther development of the compound of Formula I.

Example 8 Pharmacokinetics and Brain Penetration of the Compound ofFormula I in Rats

In Example 11, the pharmacokinetics of the compound of Formula I wasevaluated following single intravenous, intraperitoneal, and oraladministration to rats to compare the oral bioavailability of twodifferent formulations and to measure the brain penetration afterintravenous administration.

Dosage Forms and Administration

A solution of the compound of Formula I (4.7 mg/mL) in water and asuspension of the compound of Formula I (5 mg/mL) in SSV (standardsuspension vehicle) were prepared. The compound of Formula I (dosesexpressed as free base) was administered to rats as set forth in Table15.

TABLE 15 Formula I Dosage Forms and Administration. Dose Rat No.Protocol Route Formulation (mg/kg) NJ418-423/98 192/98 Lt i.p., p.o.,i.v. Solution in water 9.4* NJ672-679/98 193/98 Lt i.v. Solution inwater 9.4* NJ97-98/99 43/99 Sp p.o. Suspension in SSV 10 NJ99-102/9943/99 Sp p.o., i.v. Solution in water 9.4 *Corresponding to 10 mg/kgFormula I

Test Animals

The experiments were performed in 20 male rats (Strain RoRoFuellinsdorf, body weight 230-290 g) from Biological ResearchLaboratories, Fuellinsdorf, Switzerland. The animals were housed understandard laboratory conditions throughout the study period. After anacclimatization period of 3 days, the rats were implanted with chronicjugular catheters under pentobarbital anesthesia. After surgery the ratswere on recovery for 2 days before dosing. They had free access to waterand food during the experiment.

Sample Collection

For the pharmacokinetic studies, 0.4 mL blood samples were collected atdifferent time points, up to 72 hours post-dose, from the jugular veinof the rats via catheter. For the study of brain and CSF penetration, 2mL blood samples as well as CSF and brain were collected between 0.3 and2 hours post-dose from one rat at each time point. Collection tubescontained EDTA/NAF as anticoagulant and stabilizer, respectively. Aftercentrifugation, plasma was removed. Plasma, CSF and brain samples werestored deep-frozen at approximately −20° C. until analysis using aspecific LC-MS method.

Plasma Sample Preparation

Aliquots of plasma samples of 40 μL were mixed with 50 μL buffer pH 9,50 μL of a Formula I internal standard (1 μg/mL 1-chlorobutane) and 250μL butylacetate and then shaken for 10 min. After centrifugation, 200 μLof the supernatant were evaporated to dryness at 45° C. under a streamof nitrogen. The residue was reconstituted with 200 μL acetonitrile/1%formic acid in water (30/70, v/v). An aliquot of 30 μL was injected ontoan analytical column (Waters, Symmetry C8, 2.1×150 mm, 5 μm).

Brain and CSF Sample Preparation

The frozen half brain was weighed. After thawing, the tissue wassuspended in a 2 mL eppendorf polypropylene tube with 2 volumes ofsterile apyrogen and cold NaCl (+4° C.; 0.9% solution) (0.333 g brain/mLNaCl). The tissue was homogenized (ice bath) using a Vibra-Cellultrasonic processor (Sonics & Material, Inc. Danbury, Conn.—USA) for2×10 s (amplitude: 60, energy: 25). An aliquot of the resulting brainhomogenate (40 μL) was used for extraction as described for plasma. 50μL CSF with 50 μL plasma were used for extraction as described underplasma sample preparation. Separation occurred by gradient elution usinga solvent A (acetonitrile) and a solvent B (1% formic acid in water).The flow rate was 0.3 mL/min and the gradient elution was:

Time Mobile Phase Mobile Phase (min) A (%) B (%) 0 30 70 5 60 40 9 60 4010 30 70

The sample passed to the ion-spray interface of the single quadrupolemass spectrometer. Selected ion monitoring mode (SIM) was used for massspectrometric detection. Quantification was based on peak area ratiosand calibration curve established by weighted (1/x²) linear regression.The calibration curve was established between 5 and 5000 ng/mL using dogplasma as matrix. Data acquisition and integration of SIM chromatogramswere performed using MacQuan (version 1.6) from Perkin-Elmer Sciex.

Kinetic Analysis

The pharmacokinetic parameters were estimated by non-compartmentalanalysis, using the pharmacokinetic evaluation program WinNOnlin [1].AUC(0-inf.) was calculated applying the linear trapezoidal rule andextrapolation to infinity using the apparent elimination rate constantλz and the calculated concentration at the last measurable time point.AUClast values were calculated by linear trapezoidal rule from time zeroto time of last measurable time point. Cmax, C(t), and Tmax weredetermined directly from the plasma concentration-time profiles. Theapparent terminal half-life (T_(1/2)) was derived from the equation:T_(1/2)=ln 2/λz. Means of the half-life were calculated by harmonicmeans. Plasma clearance, CL, was calculated as D/AUC(0-inf.). Volume ofdistribution, Vz, was calculated as CL/λz. The absolute bioavailabilitywas calculated from plasma concentration data as follows:

${F(\%)} = {\frac{{{AUC}\left( {0 - \infty} \right)}_{p.o.}}{{{AUC}\left( {0 - \infty} \right)}_{i.v}}*\frac{D_{i.v.}}{D_{p.o.}}*100}$

Possible small deviations of the reported mean values from thosecalculated from non-rounded pharmacokinetic parameters are due to therounding procedure of individual values. Apart from calculating meanvalues, no formal statistical analysis was performed because of the lownumber of animals.

Assay Performance

The performance of the LC-MS assay was assessed from the analysis ofcontrol samples which were measured alongside unknown samples. Theaverage inter-assay precision was 7.3% for rat plasma and 1.9% for dogplasma in the concentration range of 5-2000 ng/mL. The correspondinginter-assay inaccuracy averaged 2.7% for rat plasma, 5.3% for dog plasmaand 5.0% for brain samples. The quantification limit was set to 4 ng/mL(20% below the lowest calibration point). This was considered to beadequate to reach the objective of the study.

Plasma Concentrations of the Compound of Formula I and DerivedPharmacokinetic Parameters

The plasma concentration-time curves of the compound of Formula Ifollowing intravenous or oral administration to rats are shown in Tables16-17.

The calculated pharmacokinetic parameters are compiled in Tables 16-17.

TABLE 16 Pharmacokinetics of Formula I Following Single Oral,Intraperitoneal and Intravenous Administration of Formula I (Free Base)in Water (9.4 mg/kg Free Base) to Rats. Formulation Admin. Route FormulaI Formula I Formula I (water, p.o) (water, i.p.) (water, i.v.) Rat No.NJ418 NJ419 NJ420 NJ421 NJ422 NJ423 Cmax 991 682 1610 1730 — — (ng/mL)Tmax (h) 2 4 0.25 0.25 — — AUC (0-24 h) 15000 12600 26800 19900 2920024400 (ng · h/mL) CL* — — — — 5.38 6.44 (mL/min/kg) T_(1/2) (h) 17.718.9 106 28.1 35.9 12.5 Vz* (L/kg) — — — — 16.7 6.97 F (%) 56.0 47.0 10074.3 — — *Calculated with AUC (0-24 h)

TABLE 17 Pharmacokinetics of Formula I Following Single OralAdministration of Formula I (Hydrochloride Salt) in SSV, and Single Oraland Intravenous Administration of Formula I (Hydrochloride Salt) (9.3mg/kg Free Base) in Water to Rats. Formulation Admin. Route Formula IFormula I Formula I (HCl salt) (HCl salt) (HCl salt) (water, p.o)(water, i.p.) (water, i.v.) Rat No. NJ97 NJ98 NJ99 NJ100 NJ101 NJ102Cmax 616 796 933 1060 — — (ng/mL) Tmax (h) 2.2 4.0 6.3 4.0 — — AUC (0-48h) 12000 15200 26200 20100 31700     21400     (ng · h/mL) AUC (0-Inf.h) 12800 15800 37900 34000 49300**    32300**    (ng · h/mL) CL* — — — —4.96 7.35 (mL/min/kg) T_(1/2) (h) 11.6 10.2 26.5 27.1  25.5***  18.2***Vz* (L/kg) — — — — 11.0  11.5  F (%) 42.4 53.7 98.6 75.7 — — *Calculatedwith AUC (0-24 h); **Calculated with AUC (0-72 h); ***Without 72 hconcentration

Intravenous Administration

A solution of Formula I (hydrochloride salt) was administeredintravenously at a dose of 9.4 mg/kg to 2 male rats in two subsequentexperiments. In both studies, blood samples were obtained at 0.083,0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous application and inaddition at 48 and 72 h in the study.

Following intravenous application, the plasma concentrations showed ashort distribution phase followed by a slow decline with a mean apparentterminal half-life of 19.8 h (range 12.5 to 35.9 h, n=4). This longelimination half-life is in line with a low systemic clearance of thetest compound (5 to 7 mL/min/kg).

The volume of distribution of Formula I (12 L/kg), much higher than thetotal body water space in rats, suggested a high extravasculardistribution. These data were confirmed during a pilot whole bodyautoradiography study in rats, which showed an extensive distribution ofthe labeled compound and/or metabolites as well as a very slowelimination from the body.

In the experiment, the plasma concentration values measured at 72 hfollowing dosing were excluded from the evaluation. Plasmaconcentrations at 72 h were 4 times higher than those at 48 h. Noexplanation could be found so far for this observation. Due to thisirregular pharmacokinetic behavior with rising plasma concentrations,clearance and volume of distribution were calculated with an AUC from 0to 24 h for 2 rats and from 0 to 48 h for 2 other animals.

During the course of the studies, no overt pharmacological ortoxicological signs were observed in the rats.

Intraperitoneal and Oral Administration to Rats

Two different formulations were administered to rats: (1) a solution ofFormula I (hydrochloride salt) was administered intraperitoneally to 2male rats or orally (gavage) to 4 male rats at a dose of 9.4 mg/kg; and(2) a suspension of Formula I (free base) in SSV (standard suspensionvehicle) was administered orally at a dose of 10 mg/kg to 2 male rats.

Blood samples were obtained at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 hafter administration and in addition at 48 and 72 h.

After intraperitoneal application of the solution, the bioavailabilityof Formula I was 100 and 74% in the 2 rats. Peak concentrations werereached rapidly, within 0.25 h following dosing. The test compound hadan apparent elimination half-life of 106 and 28.1 h, respectively, inthe 2 animals.

After oral application of a solution of Formula I (hydrochloride salt)in water to 4 rats, Cmax values ranged from 682 to 1060 ng/mL and wereachieved between 2 and 6 hours after dosing. The mean (±SD) apparentterminal half-life of 13.6 h (±4.9 h) is consistent with the mean valuesfound after i.v. administration (19.8 h±10 h). The oral bioavailabilityranged from 47 to 98.6% calculated with AUC 0-24 h for protocol 192/98Ltand with AUC 0-48 h for protocol 43/99Sp.

Oral application of a suspension of Formula I (hydrochloride salt) inSSV was compared to oral application of Formula I (hydrochloride salt)in water. After application of free base, maximum plasma concentrationswere 616 and 796 ng/mL and were achieved at 2.2 and 4 h, respectively,after dosing. They were lower than peak concentrations reached with thesolution of Formula I (hydrochloride salt). The bioavailability of thisoral suspension ranged from 42 to 54% (calculated with AUC 0-48 h).

At 72 h post-dose, plasma levels were significantly higher than at 48 h.With the suspension of Formula I (hydrochloride salt) in SSV, theincrease was approximately 5-fold. With the solution of Formula I(hydrochloride salt) in water, the increase was very small. The reasonsfor these findings is unknown.

During the course of the studies, no overt pharmacological ortoxicological signs were observed in the rats.

Brain Concentrations

Brains were taken at approximately 0.25, 0.5, 1 and 2 h afterintravenous administration of Formula I into the tail vein of 8 rats (2rats per time point). For all time points, the concentrations werehigher in the brain than in the plasma, with ratios of brainhomogenate/plasma concentrations of 2.4 to 4.9. The CSF was taken at thesame time points and analyzed but the concentrations were low (4.9 to 16ng/mL or below 4 ng/mL), possibly due to the high plasma proteinbinding. This result was confirmed by the determination of the bindingof Formula I to rat plasma proteins which was 99.8%.

Conclusions

The pharmacokinetics of Formula I was assessed in the rat. The resultsindicated a long terminal half life of Formula I (19.5 h), in line witha low systemic clearance of the compound in rats (6 mL/min/kg). Theresults also indicate a high volume of distribution (12 L/kg) indicatinga pronounced extravascular distribution of the compound. A penetrationof the compound of Formula I into the brain was also observed, asindicated by brain/plasma ratios from 2.4 to 4.9 within 2 h followingi.v. administration. The results also indicated an oral bioavailabilityof Formula I (hydrochloride salt) administered in water ranged from 47to 100% (n=4). The bioavailability of Formula I (free base) in SSV was42 and 54%. The free base was considered as suitable for furtherdevelopment of the compound of Formula I.

1-15. (canceled) 16) A crystalline free-base form of the compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidehaving an X-ray powder diffraction pattern comprising a characteristicpeak in terms of 20 at 4.50±0.2°. 17) A crystalline free-base form ofthe compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidehaving an X-ray powder diffraction pattern comprising a characteristicpeak in terms of 20 at 11.50±0.2°. 18) A crystalline free-base form ofthe compound2-(3,5-bis(trifluoromethyl)phenyl)-N,2-dimethyl-N-(6-(4-methylpiperazin-1-yl)-4-(o-tolyl)pyridin-3-yl)propanamidehaving an X-ray powder diffraction pattern comprising a characteristicpeak in terms of 20 at 13.10°±0.2°. 19) The crystalline form of claim16, having an X-ray powder diffraction pattern further comprising thefollowing characteristic peak, in terms of 2θ: 11.5°±0.2°. 20) Thecrystalline form of claim 16, having an X-ray powder diffraction patternfurther comprising the following characteristic peak, in terms of 2θ:13.1°±0.2°. 21) The crystalline form of claim 16, having an X-ray powderdiffraction pattern further comprising the following characteristicpeaks, in terms of 2θ: 11.5°±0.2°; and 13.10°±0.2°. 22) The crystallineform of claim 17, having an X-ray powder diffraction pattern furthercomprising the following characteristic peaks, in terms of 2θ:13.1°±0.2°. 23) The crystalline form of claim 16 in a micronized state.24) The crystalline form of claim 17 in a micronized state. 25) Thecrystalline form of claim 18 in a micronized state. 26) The crystallineform of claim 19 in a micronized state. 27) The crystalline form ofclaim 20 in a micronized state. 28) The crystalline form of claim 21 ina micronized state. 29) The crystalline form of claim 22 in a micronizedstate. 30) The crystalline form of claim 16 in the form of a hydratehaving a degree of hydration less than
 3. 31) The crystalline form ofclaim 17 in the form of a hydrate having a degree of hydration less than3. 32) The crystalline form of claim 18 in the form of a hydrate havinga degree of hydration less than
 3. 33) The crystalline form of claim 23in the form of a hydrate having a degree of hydration less than
 3. 34)The crystalline form of claim 24 in the form of a hydrate having adegree of hydration less than
 3. 35) The crystalline form of claim 25 inthe form of a hydrate having a degree of hydration less than
 3. 36) Thecrystalline form of claim 16 in the form of an anhydrate. 37) Thecrystalline form of claim 17 in the form of an anhydrate. 38) Thecrystalline form of claim 18 in the form of an anhydrate.